Abstract Inhibitors, alloantibodies against factor VIII, are the most significant complication of factor VIII replacement therapy for hemophilia A. The development of inhibitors substantially increases the mortality and morbidity of the disease. Unfortunately, current therapeutic options are limited. New therapies are urgently needed to address these unmet clinical needs of hemophilia A patients with inhibitors. To this end, we propose to several new strategies with translational potential to address this clinical problem, but will also delineate the pathological immune response responsible for inhibitor development and persistence. Together, our laboratories have extensive expertise in hemophilia and cellular therapy. Using hemophilia models, we seek to develop novel therapeutic approaches based on recent success of chimeric antigen receptor (CAR) technology for B cell neoplasia. We are harnessing the effect of CAR T cell on B cells to specifically eliminate those involved in generation of immune response to FVIII protein. In aim 1, our goal is to eradicate inhibitors in murine models by leveraging the proven the ability of CAR T-cells to eliminate specific compartments in the B- cell lineage. Specifically, we will target memory B-cells and plasma cells through their CD19 and B-cell maturation antigen (BCMA) respectively. This strategy will provide an ideal experimental system to define the contribution of specific populations of B-cells or plasma cells responsible for alloantibody production and persistence. Because inhibitor eradication does not necessarily induce immune tolerance, in aim 2, our goal is to test the ability of these CAR T-cells to induce immune tolerance. In this aim, we will determine if the reconstitution immunological environment created after B- or plasma-cell depletion is biased towards inducing tolerance. We will test this hypothesis in both nave and inhibitor mice. This study will also clarify the immune cells responsible for amnestic or new alloantibody responses. Because of the potential safety concern related to broad B- and plasma-cell deletion leading to immunosuppression, we have also developed technology that specifically targets anti-FVIII immune cells while sparing non-pathological cells. We have accomplished this by reversing the traditional CAR design and developing a novel construct, which we have called a chimeric allo- antigen receptor (or CALLAR), were domains of FVIII are coupled to modified T-cell receptors. In aim 3, we test the safety, efficacy, and specificity of CALLAR T-cells to eradicate inhibitors in murine models while avoiding non-pathological cells. Together, we propose a comprehensive plan to characterize and identify B cells in pathological responses that are highly applicable to other disease treatments which are also complicated by the formation of antibodies to enzyme replacement therapy. Specifically, the CALLAR strategy may be applicable for diseases in which specific immune epitopes are known and thus can be personalized for a given target to be express in the reversed CAR. These data provide a new therapeutic platform for immunotherapy for benign diseases using, in some situations, therapies already available.